Therapeutic canine immunoglobulins and methods of using same

ABSTRACT

A method of preparing a canine antibody suitable for use in the therapeutic treatment of a canine is provided. In particular, there is provided immunoglobulins which can be selected for the characteristic of whether they mediate downstream complement mediated immune activation when bound to a target antigen. Canine derived antibodies comprising specific heavy chain isotypes are provided. The invention extends to the use of the immunoglobulins of the invention in methods of treating conditions such as pain, inflammatory conditions and cancerous conditions in a canine.

FIELD OF THE INVENTION

The present invention relates to canine or canine derived antibodies,which have specific heavy chain constant regions, for use as antagonistsof soluble extracellular mediators and/or cell surface receptors. Theinvention extends to the therapeutic use of the antibodies, or fragmentsthereof, in methods for the selective treatment of conditions such asinflammation, pain, cancer or infection in a canine subject.

BACKGROUND TO THE INVENTION

Recombinant immunoglobulins and fusion proteins constructed usingconstant domain fragments of immunoglobulins are used to treat manyhuman diseases including inflammatory diseases (e.g. rheumatoidarthritis, psoriasis, inflammatory bowel disease), allergies (e.g.asthma), cancers (e.g. lymphoma, breast cancer, bowel cancer),infectious diseases (e.g. RSV infection), pain (e.g. osteoarthriticpain, cancer pain, lower back pain) and eye disease (e.g. age-relatedmacular degeneration).

The molecular targets for therapy include cytokines and chemokines (e.g.interleukin-1 (IL-1), interleukin-5 (IL-5), granulocytecolony-stimulating factor (GCSF), granulocyte-macrophage colonystimulating factor), growth factors (e.g. nerve growth factor (NGF),vascular endothelial cell growth factor (VEGF), tumour necrosis factor(TNF)), cell surface receptors (e.g. HER-2, VEGFR, EGFR, CD20), cellsurface-bound growth factors (e.g. unprocessed tumour necrosis factor),viruses (e.g. RSV) and components of the complement cascade (e.g. C5).Many other targets that have evidence for involvement in diseaseprocesses are known (e.g. as described in the IMGT/MAb-DB databaseVersion 1.3.1 14 Dec. 2011, (www.imgt.org/mAb-DB/query).

Native immunoglobulins are produced as different major subtypes,including immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulinM (IgM) or immunoglobulin E (IgE) and in response to infection theseimmunoglobulins play various roles in pathogen recognition by binding totarget antigens, neutralisation, destruction and removal. ImmunoglobulinG is produced as several different isotypes (also known as isoforms),such as (in humans) IgG1, IgG2, IgG3 and IgG4. These antibody isotypesvary in structure, in particular with regard to differences in the aminoacid sequences of the constant region, particularly around the hingeregion of the constant domain (Fc) between the C1 and C2 domains.

Different antibody isotypes also differ in terms of the downstreameffector functions which the antibody mediates. For example, theconstant region sequence of an antibody can mediate a strong influenceon characteristics such as effector functions (ADCC, complement fixingand activation), pharmacokinetics, and physical properties of anantibody. Antibodies having different isotypes also differ in terms oftheir ability to bind to IgG Fc receptors on immune cells. In humans,IgG1 and IgG3 are active in recruiting complement to aid in targetdestruction by the cascade of complement enzymes in the blood (CDC:complement-dependent cytotoxicity), and similarly IgG1 and IgG3 bind Fcreceptors on immune cells that target the bound antigen for destructionby antibody-mediated cellular cytotoxicity (ADCC). By contrast, IgG2 andIgG4 do not recruit complement or activate ADCC mediated attack andsimply bind to the target antigen with high affinity to inhibit orneutralise its activity.

Recombinant immunoglobulins and fusion proteins made from the same aredesigned to take into account the activity of the Fc isotype whenconsidering the target for disease intervention. For example, it ispreferable when considering a therapeutic approach which aims to useantibodies for the targeted killing of human cancer cells to constructthe recombinant immunoglobulin from IgG1 or IgG3 isotype Fc domains, asthe use of these isotypes will drive immune mediated destructivemechanisms such as CDC and ADCC. By contrast, when targeting solublemediators in the context of sensitive human tissues, the Fc domain iseither omitted (e.g. in treatment of human age-related maculardegeneration Fab fragments targeting VEGF are preferred), or isconstructed using IgG2 or IgG4 Fc domains (e.g. targeting nerve growthfactor in the context of neuropathic or inflammatory pain, or complementC5 in nephritis, psoriasis or rheumatoid arthritis). Theseconsiderations also apply to immunoglobulin fusion proteins, such assoluble TNF receptor Fc fusion proteins in the treatment of conditionssuch as rheumatoid arthritis, which are based on human IgG1 Fctherapeutics.

In canines and other species such as mice and horses, immunoglobulinisoforms also exist but have insufficient homology between one anotherto determine a priori which sequence will be active or inactive ininducing downstream effector functions such as CDC or ADCC. Furthermore,the number of immunoglobulins varies between species (e.g. in dog thereare four IgG immunoglobulins, these being defined as calgG-A, calgG-B,calgG-C, and calgG-D (Tang et al., 2001). In horses, there are seven IgGisotypes (Wagner, 2006).

It is not possible to determine from sequence analysis or sequencehomology alone whether a specific immunoglobulin isotype of a non-humanspecies will be active or inactive in terms of mediating Fc receptorbinding and downstream effector function. However, if these were known,it would be of significant value as the choice of isotype constantregions for antibody generation can be critical in order to provide thetherapeutic effectiveness of an antibody or antibody based therapeutics,such as an antibody binding fragment or fusion protein.

SUMMARY OF THE INVENTION

Following extensive experimentation, it has been surprisingly identifiedby the present inventor that the isotypes of canine IgG immunoglobulinshare the characteristics observed in human IgG antibodies that certainIgG antibody isotypes are active in terms of activating immune effectorfunctions, while other IgG antibody isotypes do not activate immuneeffector functions and are accordingly inactive. Furthermore, of thefour known canine heavy chain immunoglobulins (known as HCA (calgG-A),HCB (calgG-B), HCC (calgG-C) and HCD (calgG-D)), the inventor hassurprisingly identified that heavy chain constant domains from two(calgG-B and calgG-C) of the four canine heavy chain immunoglobulins,when constructed as various recombinant forms targeting differenttherapeutic targets, surprisingly bind complement, whereas the other two(calgG-A and calgG-D) do not.

Accordingly, the present invention defines certain recombinant canineimmunoglobulins, or fusion proteins made therefrom, which may be used inthe therapy of canines where target destruction is desired (e.g. incancer or infectious disease treatment); and certain other isoformswhich may be preferred for therapeutic treatments in canines wheretarget neutralisation alone, rather than target destruction, is desired(e.g. in the treatment of pain).

The present invention therefore provides recombinant canineimmunoglobulins that can be distinguished by their ability or otherwiseto bind to the first component of the complement cascade, based on theisotype of their heavy chain constant domain. As a result, and for thefirst time, recombinant canine immunoglobulins can be selected accordingto their intended use in treatment of disease in canines, whether forpurposes where the intended target is selected for immune mediateddestruction through complement mediated cytotoxicity (CDC; e.g. for usein killing canine tumours in vivo) or where the target is selectedsimply for neutralisation in the absence of undesirable immune mediateddestruction (e.g. in the proximity of nerves, or in the eye).

According to a first aspect of the invention there is provided anantibody, fusion protein or a binding fragment thereof for use in thetherapeutic treatment of a canine, wherein said antibody, fusion proteinor binding fragment has a heavy chain constant domain comprising theamino acid sequence of SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:13,wherein the amino acid sequence of the heavy chain minimises theactivation of downstream immune system effector functions when theantibody, fusion protein or binding fragment is bound to its targetantigen.

In certain embodiments, the therapeutic treatment of the canine relatesto the treatment of pain or inflammation or a condition associatedtherewith, such as arthritis or an arthritic condition.

A yet further aspect of the invention provides use of an antibody,fusion protein or a binding fragment thereof comprising a heavy chainconstant domain having the amino acid sequence of SEQ ID NO:8, SEQ IDNO:11 or SEQ ID NO:13, wherein the amino acid sequence of the heavychain minimises the activation of downstream immune system effectorfunctions when the antibody, fusion protein or binding fragment is boundto its target antigen, in the preparation of a medicament for use in thetreatment of pain or inflammation in a canine subject or a conditionassociated therewith, such as arthritis or an arthritic condition.

A yet further aspect of the present invention provides a method fortreating, inhibiting or ameliorating pain or inflammation or a conditionassociated therewith, such as arthritis or an arthritic condition, in acanine subject in need thereof, the method comprising the steps of:

-   -   providing an antibody, fusion protein or a binding fragment        thereof which binds specifically to a target antigen which has a        specific function in the treatment or prevention of pain or        inflammation, wherein the antibody, fusion protein or binding        fragment thereof has a heavy chain constant domain comprising        the amino acid sequence of SEQ ID NO:8, SEQ ID NO:11 or SEQ ID        NO:13 and wherein the antibody, fusion protein or binding        fragment thereof does not activate downstream immune system        effector functions, and    -   administering a therapeutically effective amount of the        antibody, fusion protein or binding fragment thereof to the        canine subject.

A yet further aspect of the present invention provides a canine derivedantibody, fusion protein or a binding fragment thereof which has a heavychain constant domain comprising the amino acid sequence of SEQ ID NO:8,SEQ ID NO:11 or SEQ ID NO:13 for use in the preparation of a medicamentfor the treating, inhibiting or ameliorating pain or inflammation in acanine subject.

In certain embodiments, the pain is neuropathic pain. In particular, thepain may be peri-operative, post-operative or post-surgical pain.Post-operative pain may result following any operating procedure whichin canines may include, but is not limited to orthopaedic surgery, softtissue surgery, ovariohysterectomy procedures, castration procedures andthe like. In certain further embodiments, the pain is chronic painassociated with cancer or a cancerous condition (oncologic pain). Incertain further embodiments, the pain is associated with, or resultingfrom rheumatoid arthritis, osteoarthritis, inflammation or pruritis.

A yet further aspect of the present invention provides a method for thetreatment of arthritis or an arthritic condition in a canine subject,the method comprising the steps of:

-   -   providing an antibody, fusion protein or a binding fragment        thereof which binds specifically to a target antigen which has a        specific function in the treatment or prevention of pain or        inflammation, wherein the antibody, fusion protein or binding        fragment thereof has a heavy chain constant domain comprising        the amino acid sequence of SEQ ID NO:8, SEQ ID NO:11 or SEQ ID        NO:13 and wherein the antibody, fusion protein or binding        fragment thereof does not activate downstream immune system        effector functions, and    -   administering a therapeutically effective amount of the        antibody, fusion protein or binding fragment thereof to the        canine subject in need of such treatment.

In certain embodiments, the foregoing method of the invention furthercomprises the step of co-administering at least one further agent whichmay enhance and/or complement the effectiveness of the antibody of theinvention. For example, the antibody or antigen binding fragment thereofmay be co-administered along with at least one analgesic, NSAID, opioid,corticosteroid or steroid. Examples of suitable analgesics include, butare not limited to butorphanol, 10 buprenorphine, fentanyl, flunixinmeglumine, merpidine, morphine, nalbuphine and derivatives thereof.Suitable NSAIDS include, but are not limited to acetaminophen,acetylsalicylic acid, carprofen, etodolac, ketoprofen, meloxicam,firocoxib, robenacoxib, deracoxib and the like.

In certain embodiments, the foregoing methods may be accompanied by theadministration of at least one further agent. Said agent may be atherapeutically active agent which may be one or more of the groupselected from: an antibiotic, antifungal, antiprotozoal, antiviral orsimilar therapeutic agents. Furthermore the at least one further agentmay be an inhibitor of mediator(s) of inflammation such as aPGE-receptor antagonist, an immunosuppressive agent, such ascyclosporine, an anti-inflammatory glucocorticoids. In certain furtheraspects the at least one further agent may be an agent which is used forthe treatment of cognitive dysfunction or impairment, such as memoryloss or related conditions which may become increasingly prevalent inolder canines. Further still, the at least one further agent may be ananti-hypertensive or other compound used for the treatment ofcardiovascular dysfunction, for example to treat hypertension,myocardial ischemia, congestive heart failure and the like. Furtherstill, the at least one further agent may be a diuretic, vasodilator,beta-adrenergic receptor antagonist, angiotensin-II converting enzymeinhibitor, calcium channel blocker and HMG-CoA reductase inhibitor.

In certain embodiments of the foregoing aspects of the invention, thedownstream immune system effector functions are selected from the groupcomprising complement dependent cytotoxicity (CDC), antibody dependentcell mediated cytotoxicity (ADCC), and antibody dependent cellularpathogenesis (ADCP). In specific embodiments, the amino acid sequence ofthe heavy chain constant domain inhibits binding of the heavy chain toC1q, this preventing induction of the complement cascade and complementdependent cytotoxicity (CDC).

In certain embodiments, the target antigen is a soluble mediator. Incertain embodiments, the target antigen is nerve growth factor (NGF). Incertain embodiments, the antibody specifically binds to and antagonisesa receptor which mediates pain or inflammation. In certain furtherembodiments, the target antigen can be selected from the groupconsisting of, but not limited to: cytokines or chemokines (e.g.interleukin-1 and related interleukins IL-2 through IL-35, granulocytecolony-stimulating factor, granulocyte-macrophage colony stimulatingfactor, erythropoietin, thrombopoetin, leukaemia inhibitory factor,ciliary neurotrophic factor, oncostatin M), growth factors (e.g. nervegrowth factor (NGF), brain-derived neurotrophic factor (BDNF),neurotrophin-3, neurotrophin-4, vascular endothelial cell growth factor(VEGF), tumour necrosis factor (TNF), cell surface receptors (e.g.HER-2, VEGFR, EGFR, CD20), cell surface-bound growth factors (e.g.unprocessed tumour necrosis factor), viruses (e.g. RSV) and componentsof the complement cascade (e.g. C5, C5a).

According to yet further aspect of the invention there is provided anantibody, fusion protein or a binding fragment thereof for use in thetherapeutic treatment of a canine, wherein said antibody, fusion proteinor binding fragment has a heavy chain constant domain comprising theamino acid sequence of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:14wherein the amino acid sequence of the heavy chain mediates theactivation of downstream immune system effector functions when theantibody, fusion protein or binding fragment is bound to its targetantigen.

In certain embodiments, the therapeutic treatment of the canine relatesto the treatment of a cancerous or malignant condition.

A yet further aspect of the invention provides use of an antibody,fusion protein or a binding fragment thereof comprising a heavy chainconstant domain having the amino acid sequence of SEQ ID NO:9, SEQ IDNO:10 or SEQ ID NO:14, wherein the amino acid sequence of the heavychain mediates the activation of downstream immune system effectorfunctions when the antibody, fusion protein or binding fragment is boundto its target antigen, in the preparation of a medicament for use in thetreatment of a cancerous or malignant condition in a canine subject.

A yet further aspect of the present invention provides a method for thetreatment or prevention of a cancerous or malignant condition in acanine subject, the method comprising the steps of:

-   -   providing an antibody, fusion protein or a binding fragment        thereof which binds specifically to a target antigen which has a        specific function in the treatment of a cancerous or malignant        condition, wherein the antibody, fusion protein or binding        fragment has a heavy chain constant domain comprising the amino        acid sequence of SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:14 and        wherein the antibody, fusion protein or binding fragment        activates downstream immune system effector functions, and    -   administering a therapeutically effective amount of the        antibody, fusion protein or binding fragment to the canine        subject in need of such treatment.

In certain embodiments, the foregoing method of the invention furthercomprises the step of co-administering at least one further agent whichmay enhance and/or complement the effectiveness of the antibody of theinvention.

A yet further aspect of the present invention provides use of a caninederived antibody, fusion protein or a binding fragment thereof, whichhas a heavy chain constant domain comprising the amino acid sequence ofSEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:14 in the preparation of amedicament for the treatment of cancerous or malignant condition in acanine subject.

In certain embodiments of the foregoing aspects of the invention, thedownstream immune system effector functions are selected from the groupcomprising complement dependent cytotoxicity (CDC), antibody dependentcell mediated cytotoxicity (ADCC), and antibody dependent cellularpathogenesis (ADCP). In specific embodiments, the amino acid sequence ofthe heavy chain constant domain provides for binding of the heavy chainto C1q, this inducing the complement cascade and complement dependentcytotoxicity (CDC). In certain embodiments, the heavy chain constantdomain provides for binding to Fc receptors, which may in turn mediateADCP and/or ADCC immune responses.

In certain embodiments, the target antigen is a cancer specific antigen.In certain further embodiments of the invention, the target antigen maybe selected from the group of membrane bound proteins expressed oncanine tumour cells. In further embodiments of the invention, themembrane bound canine tumour proteins may be selected from the group ofproteins including CD2, CD4, CD8, CD20, EGFR, VEGFR, HER2 and the like.In certain further embodiments, the target antigen can be selected fromthe group consisting of, but not limited to: cytokines and chemokines(e.g. interleukin-1 (IL-1), IL-2, IL-3 and interleukins numericallythrough to IL-35, granulocyte colony-stimulating factor,granulocyte-macrophage colony stimulating factor, erythropoietin,thrombopoetin, leukaemia inhibitory factor, ciliary neurotrophic factor,oncostatin M), growth factors (e.g. nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophin-3,neurotrophin-4, vascular endothelial cell growth factor (VEGF), tumournecrosis factor (TNF)), cell surface receptors (e.g. HER-2, VEGFR, EGFR,CD20), cell surface-bound growth factors (e.g. unprocessed tumournecrosis factor), viruses (e.g. RSV) and components of the complementcascade (e.g. C5, C5a).

A yet further aspect of the invention provides for an antibody, fusionprotein or a binding fragment thereof for use in the treatment of acondition in a canine, wherein the antibody, fusion protein or bindingfragment has a heavy chain constant domain which does not bind to C1qwhen the antibody, fusion protein or binding fragment is bound to itstarget antigen and wherein the antibody, fusion protein or bindingfragment can be purified using Protein A chromatography.

In certain embodiments, said antibody, fusion protein or bindingfragment has a heavy chain constant domain comprising the amino acidsequence of SEQ ID NO:13. In certain embodiments, said antibody, fusionprotein or binding fragment has a heavy chain constant domain selectedfrom the group consisting of SEQ ID NO:6, SEQ ID NO:12 and SEQ ID NO:15.

A yet further aspect of the invention provides use of an antibody,fusion protein or a binding fragment which comprises a heavy chainconstant domain which does not bind to C1q when the antibody, fusionprotein or binding fragment is bound to its target antigen and whereinthe antibody, fusion protein or binding fragment can be purified usingProtein A chromatography in the preparation of a medicament for thetreatment of a condition associated with pain and/or inflammation in acanine.

In certain embodiments, said antibody, fusion protein or bindingfragment has a heavy chain constant domain comprising the amino acidsequence of SEQ ID NO:13. In certain embodiments, said antibody, fusionprotein or binding fragment has a heavy chain constant domain selectedfrom the group consisting of SEQ ID NO:6, SEQ ID NO:12 and SEQ ID NO:15.

A yet further aspect of the present invention provides a method fortreating, inhibiting or ameliorating pain or inflammation or a conditionassociated therewith, such as arthritis or an arthritic condition, in acanine subject in need thereof, the method comprising the steps of:

-   -   providing an antibody, fusion protein or binding fragment which        comprises a heavy chain constant domain which does not bind to        C1q when the antibody is bound to its target antigen and wherein        the antibody can be purified using Protein A chromatography, and    -   administering a therapeutically effective amount of the        antibody, fusion protein or binding fragment thereof to the        canine subject.

In certain embodiments, said antibody, fusion protein or bindingfragment has a heavy chain constant domain comprising the amino acidsequence of SEQ ID NO:13. In certain embodiments, said antibody, fusionprotein or binding fragment has a heavy chain constant domain selectedfrom the group consisting of SEQ ID NO:6, SEQ ID NO:12 and SEQ ID NO:15.

In various further aspects, the invention extends to: (i) nucleic acidswhich encode any of the foregoing antibodies, fusion proteins orantibody fragments of the invention, (ii) vectors which carry saidnucleic acids, (iii) host cells carrying said vectors. The inventionfurther extends to methods for producing antibodies and fusion proteinsas defined in the foregoing statements of invention. In a yet furtheraspect, the present invention extends to pharmaceutical compositionswhich comprise the antibodies or fusion proteins of the presentinvention along with at least one carrier, diluent or excipient.

A further aspect of the invention provides a recombinant antibody,fusion protein or binding fragment thereof which can be therapeuticallyadministered to a canine in order to specifically bind to a targetantigen and which further mediates an immune response which ischaracterised by C1q complement binding to said antibody, fusion proteinor binding fragment thereof and associated complement dependentcytotoxicity, wherein the heavy chain of the antibody, fusion protein orbinding fragment thereof comprises canine immunoglobulin heavy chainconstant domain isotype B (HCB, calgG-B) having an amino acid sequenceof SEQ ID NO:9, canine immunoglobulin heavy chain constant domainisotype C (HCC, calgG-C) having an amino acid sequence of SEQ ID NO:10or aglycosyl canine immunoglobulin heavy chain isotype C (HCC, calgG-C)having an amino acid sequence of SEQ ID NO:14.

A yet further aspect of the present invention provides use of anantibody, fusion protein or binding fragment thereof which comprisescanine immunoglobulin heavy chain constant domain isotype B (HCB,calgG-B) having an amino acid sequence of SEQ ID NO:9, canineimmunoglobulin heavy chain constant domain isotype C (HCC, calgG-C)having an amino acid sequence of SEQ ID NO:10 or aglycosyl canineimmunoglobulin heavy chain isotype C (HCC, calgG-C) having an amino acidsequence of SEQ ID NO:14 in the preparation of a medicament for use inthe treatment of a condition where specific binding to a target antigenis required and where an immune response which is characterised by C1qcomplement binding to said antibody, fusion protein or binding fragmentthereof and associated complement dependent cytotoxicity is desirable.

A yet further aspect of the present invention provides a method for thetreatment of a cancerous condition in a canine subject, the methodcomprising the step of:

-   -   providing an immunoglobulin, fusion protein or a binding        fragment thereof which has binding specificity for a tumour        specific antigen and which further comprises canine        immunoglobulin heavy chain constant domain isotype B (HCB,        calgG-B) having an amino acid sequence of SEQ ID NO:9, canine        immunoglobulin heavy chain constant domain isotype C (HCC,        calgG-C) having an amino acid sequence of SEQ ID NO:10 or        aglycosyl canine immunoglobulin heavy chain isotype C (HCC,        calgG-C) having an amino acid sequence of SEQ ID NO:14, and    -   administering a therapeutically effective amount of the        immunoglobulin, fusion protein or binding fragment to the canine        subject in need thereof.

In various further aspects, the invention extends to antibodies orfusion proteins which bind to a desired target antigen and whichcomprise heavy chain constant domains which do not bind C1q and whichaccordingly do not mediate an immune response involving complementdependent cytotoxicity.

Accordingly, in a yet further aspect of the present invention, there isprovided a recombinant antibody, fusion protein or binding fragmentthereof which can be therapeutically administered to a canine in orderto specifically bind to a target antigen, wherein the constant domain ofthe antibody or fusion protein does not bind to C1q complement andwherein the heavy chain of the constant domain comprises canineimmunoglobulin heavy chain constant domain isotype A (HCA, calgG-A)having an amino acid sequence of SEQ ID NO:8, canine immunoglobulinheavy chain constant domain isotype D (HCD, calgG-D) having an aminoacid sequence of SEQ ID NO:11 or an aglycosyl canine immunoglobulinheavy chain constant domain isotype B having an amino acid sequence ofSEQ ID NO:13 (HCB*, calgG-B).

A yet further aspect of the present invention provides use of anantibody, fusion protein or binding fragment thereof which comprisescanine immunoglobulin heavy chain constant domain isotype A (HCA,calgG-A) having an amino acid sequence of SEQ ID NO:8, canineimmunoglobulin heavy chain constant domain isotype D (HCD, calgG-D)having an amino acid sequence of SEQ ID NO:11 or an aglycosyl canineimmunoglobulin heavy chain constant domain isotype B having an aminoacid sequence of SEQ ID NO:13 (HCB*, calgG-B) in the preparation of amedicament for use in the treatment of a condition where specificbinding to a target antigen is required and where an immune responsewhich is characterised by C1q complement binding to said antibody,fusion protein or binding fragment thereof and associated complementdependent cytotoxicity is not desirable.

A yet further aspect of the present invention provides a method for thetreatment of a condition in a canine subject, the method comprising thestep of:

-   -   providing an immunoglobulin, fusion protein or a binding        fragment thereof which has binding specificity for a tumour        specific antigen and which further comprises canine        immunoglobulin heavy chain constant domain isotype A (HCA,        calgG-A) having an amino acid sequence of SEQ ID NO:8, canine        immunoglobulin heavy chain constant domain isotype D (HCD,        calgG-D) having an amino acid sequence of SEQ ID NO:11 or an        aglycosyl canine immunoglobulin heavy chain constant domain        isotype B having an amino acid sequence of SEQ ID NO:13 (HCB*,        calgG-B), and        -   administering a therapeutically effective amount of the            immunoglobulin, fusion protein or binding fragment to the            canine subject in need thereof.

In certain embodiments, the aglycosylated constant domain designed forantibody construction in the absence of CDC activity isalanine-substituted aglycosylated heavy chain HCB having an amino acidsequence of SEQ ID NO:6 (denoted HCB*). In various further aspects ofthe invention, the antibodies incorporating heavy chains HCA, HCD or CDCinactive aglycosylated forms of HCA, HCB or HCD (HCA*, HCB* or HCD*: SEQID NO:12, SEQ ID NO:13 and SEQ ID NO:15) are directed to canine growthfactors, hormones, cytokines, chemokines or other soluble mediators suchas components of the complement cascade. In certain embodiments, theantibodies incorporating heavy chains HCA, HCD or HCB* are directed tocanine nerve growth factor (NGF) for the purposes of neutralising canineNGF biological activity in a canine, without inducing CDC.

In certain embodiments of the foregoing aspects of the invention theantibody is a monoclonal antibody. In certain further embodiments, theantibody is a chimeric antibody. In some embodiments, the antibody is acaninised antibody, that is, an antibody which has an amino acidsequence which has been de-immunised such that neutralising antibodieswill not be produced there against when administered to a caninesubject. Typically the heavy chain constant domains of the antibody areselected or modified by way of amino acid substitution or deletion suchthat the constant domains do not mediate downstream effector functions.In certain embodiments, the antibody may be conjugated to at least onereporter molecule.

In certain further embodiments at least one residue in the constantdomain of the antibodies or fusion proteins of the foregoing aspects ofthe invention can be substituted or deleted in order to prevent theglycosylation of that residue. In a further aspect of the invention thecanine immunoglobulin heavy chain constant domain may be fused whole orin part to the extracellular domain of a cytokine or chemokine receptoror other trans-membrane protein (e.g. the TNF receptor), whereby thewhole or fragment of the canine immunoglobulin heavy chain constantdomain is selected from the group HCA, HCD or HCB* where it is desiredthat the canine extracellular domain-immunoglobulin heavy chain fusionprotein does not activate CDC (e.g. where TNF receptor Fc fusionproteins are designed for the neutralization of soluble TNF) orconversely, the canine immunoglobulin heavy chain constant domain isselected from the group HCB, HCC or HCC* where it is desired that thecanine extracellular domain-immunoglobulin heavy chain fusion protein(e.g. where TNF receptor Fc fusion proteins are designed to killmembrane-associated TNF bearing inflammatory cells).

In further aspects of the invention, the canine receptor-Fc fusionproteins may be selected from the group of extracellular domains ofmembrane bound receptors found on canine cells fused to canineimmunoglobulin domain heavy chain Fc regions. In a further aspect of theinvention, the antibodies incorporating heavy chains HCB, HCC or HCC*are directed to CD20 for the purposes of inducing CDC of canine CD20expressing cells, such as canine lymphoma cells through the binding ofthese antibodies to CD20 on their surface.

Furthermore, it is preferred that the caninised antibodies are notcross-reactive to any other epitopes present in canines, and furtherthat neutralising antibodies are not generated against the antibodies ofthe invention when they are administered to a canine.

In certain further embodiments, modifications to the amino acid sequenceof the constant regions of the heavy chain may be made to the antibodiesor fusion proteins of the invention. Said modification may involve theaddition, substitution or deletion of one or more amino acid residues.Said amino acid changes are typically performed in order to modify thefunctional characteristics of the antibody. For example, amino acidmodification may be performed to prevent downstream effector functionsmediated by the antibody constant domains, for example by preventing theability of the antibody to bind to Fc receptors, activate complement orinduce ADCC. Furthermore, modifications may be made to the amino acidresidues of the hinge region of the heavy chain constant domain in orderto modify the circulatory half-life of an antibody when it isadministered to a canine.

In some embodiments, the invention provides multi-specific ormultivalent antibodies comprising an antibody or binding fragment of theinvention coupled or conjoined to other antibodies with differentbinding specificities for use in combination therapy. A multi-specificantibody comprises at least one antibody or binding fragment specific toa first epitope, and at least one binding site specific to anotherepitope present on the antigen, or to a different antigen. A multivalentantibody comprises antibodies or antibody binding fragments which havebinding specificity to the same epitope. Accordingly, in certainembodiments, the invention extends to an antibody fusion proteincomprising four or more Fv regions or Fab regions of the antibodies ofthe present invention. In certain further embodiments, the inventionextends to a bispecific antibody, wherein an antibody or bindingfragment thereof according to the present invention is linked to asecond antibody or binding fragment thereof which has binding specificfor a second target, said target not being the first antigen. Suchmultivalent, bispecific or multispecific antibodies can be made by avariety of recombinant methods which would be well known to the personskilled in the art.

In certain embodiments, the antibody, fusion protein or antigen bindingfragment is administered to the canine as part of the foregoing methodsat a dose ranging from about 0.01 mg/kg of body weight to about 10 mg/kgof body weight, in particular from 0.03 mg/kg of body weight to about 3mg/kg of body weight.

In various further aspects, the present invention extends to acomposition comprising an antibody, fusion protein or binding fragmentthereof according to any foregoing aspect of the invention. In certainembodiments, the composition further comprises at least onepharmaceutically acceptable carrier. In certain embodiments, thecomposition may further comprise at least one analgesic, NSAID, opioid,corticosteroid or steroid.

In various further aspects, the present invention extends to isolatednucleic acid which encodes the antibody, fusion protein or antibodybinding fragments of the invention. Accordingly, a yet further aspect ofthe invention provides an isolated nucleic acid that encodes anantibody, fusion protein or antigen-binding fragment according to any ofthe foregoing aspects of the invention. In certain embodiments, theisolated nucleic acid further encodes one or more regulatory sequencesoperably linked thereto.

In a further aspect there is provided an expression vector comprising apolynucleotide encoding a heavy and/or light chain variable domain or aheavy and/or light chain constant domain of the invention. In certainembodiments the expression vector further comprises one or moreregulatory sequences. In certain embodiments the vector is a plasmid ora retroviral vector. A yet further aspect provides a host cellincorporating the expression vector of the foregoing aspect of theinvention. A further aspect of the invention provides a host cell whichproduces the antibody of any of the foregoing aspects of the invention.

A yet further aspect of the invention provides a method for producing anantibody or fusion protein of the invention, the method comprising thestep of culturing the host cell of the foregoing aspect of the inventionto allow the cell to express the antibody. A yet further aspect of thepresent invention provides a method of producing the antibody or fusionprotein of the invention comprising the steps of expressing one or moreof the polynucleotides/nucleic acids or vectors of the foregoing aspectsof the invention which express the light and/or heavy chains of theantibodies of the invention in a suitable host cell, recovering theexpressed polypeptides, which may be expressed together in a host cell,or separately in different host cells, and isolating antibodies. A yetfurther aspect of the invention provides a method for treating,ameliorating or inhibiting pain in a canine, the method comprising thestep of administering to the canine an effective amount of apolynucleotide which encodes an antibody or fusion protein having aheavy chain constant domain comprising the amino acid sequence of SEQ IDNO:8-SEQ ID NO:11.

Canine Antibody Purification

In the case of those canine antibodies where target neutralisation isdesired in the absence of unwanted immune effector activity, the presentinventor has surprisingly discovered that native isoforms of canineheavy chains that lack CDC activity also bind Staphylococcus Protein Avery poorly, if at all, and consequently this common method forpurification of antibodies cannot be used in manufacture. The inventordescribes three ways of overcoming this restriction, through the use ofalternative purification strategies and through mutation of the heavychain to prevent glycosylation during production. Purified antibodiesprepared by these methods were produced by the inventor and shown tohave the desirable properties of being safe and effective in-vivo indogs without unwanted immunogenicity.

A yet further aspect of the invention provides a method for thepurification of a canine derived immunoglobulin or an immunoglobulin orfusion protein comprising a canine heavy chain constant domain ofisotype A (HCA, calgG-A) having an amino acid sequence of SEQ ID NO:8 ora canine immunoglobulin heavy chain constant domain of isotype D (HCD,calgG-D) having an amino acid sequence of SEQ ID NO:11 from a sourcemixture, the method comprising the steps of:

-   -   (i) providing a source mixture comprising target immunoglobulins        or fusion proteins,    -   (ii) subjecting the source mixture to anion exchange        chromatography;    -   (iii) subjecting the source mixture to hydrophobic interaction        chromatography; and    -   (iv) subjecting the source mixture to size exclusion        chromatography.

In certain embodiments, the method comprises the step of buffer exchangein phosphate buffered saline. Typically the method produces a purifiedantibody which is fractionated to high purity and bioactivity.

A further aspect of the present invention provides for the method ofproduction of an aglycosylated canine antibody that can be purified byProtein A chromatography.

In various further aspects of the invention, there is provided a canineor canine derived antibody or fusion protein produced in accordance withany of the methods defined herein, for use in the therapeutic treatmentof a canine. In various further aspects, there is provided the use of ananti-canine NGF antibody in the preparation of a medicament for use intreating an immune mediated condition, or a condition associated withpain, in a canine.

A yet further aspect of the invention provides a method for thepurification of a canine derived immunoglobulin or an immunoglobulin orfusion protein comprising a canine heavy chain constant domain ofisotype A (HCA, calgG-A) having an amino acid sequence of SEQ ID NO:8 ora canine immunoglobulin heavy chain constant domain of isotype D (HCD,calgG-D) having an amino acid sequence of SEQ ID NO:11 from a sourcemixture, the method comprising the steps of:

-   -   (i) providing a source mixture comprising target        immunoglobulins,    -   (ii) subjecting the source mixture to captoadhere affinity        chromatography; and    -   (iii) subjecting the source mixture to anion exchange        chromatography.

A yet further aspect of the present invention extends to an antibody orfusion protein produced from the purification method of the foregoingaspect of the invention for use in the treatment of a canine.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show equivalent binding of anti-NGF antibodies(expressed into the supernatant of transfected CHO cells) constructedusing four different isotypes of canine heavy chains (HCA, HCB, HCC,HCD) to murine NGF by ELISA (FIG. 1A) and equivalent inhibition of NGFproliferation of TF-1 cells (FIG. 1B).

FIG. 2 shows differential binding of complement to NGF-bound anti-canineNGF antibody isotypes as measured by anti-C1q ELISA.

FIGS. 3a and 3b show binding of NGF-captured glycosylated andaglycosylated caninised anti-NGF monoclonal antibodies to complement asmeasured by anti-C1q ELISA.

FIG. 4 shows the binding of NGF-captured caninised anti-NGF monoclonalantibodies (MAbs), anti-canine VEGF MAbs and anti-human CD20/canine HCBchimeric MAb to complement measured by anti-C1q ELISA. In particular,FIGS. 4A, 4B and 4C show binding of complement to antibodies constructedusing various canine heavy chain isotypes. Caninised monoclonalantibodies (MAb) were expressed in CHO cells and tested for theirability to bind complement C1q. Panel A—caninised anti-NGF antibodies(caN-HCB, caN-HCC) compared with humanised antibody isotypes (huN-G1,huN-G4); Panel B—anti-VEGF antibodies constructed with canine HCA(caV-HCA) and HCB (caV-HCB) isotypes; Panel C—anti-CD20 antibodyconstructed using HCB isotype (mub-HCA) compared with mouse IgG2aisotype (muB-2a).

FIG. 5 shows relative recovery of anti-NGF antibody isotypes purified byProtein A and detected using anti-canine polyclonal immunoglobulin byWestern blot. The supernatants from FIG. 1 were passed over Protein Acolumns and specifically bound material eluted. Equal volumes of eluatewere subjected to SDS-PAGE. Canine isotypes HCA, HCC and HCD boundweakly to Protein A as indicated by significant material in the wash andflow through fractions. FIGS. A-D show relative recovery of canineantibody isotypes HCA, HCB, HCC and HCD by Protein A: L, load; W, wash;P, purified; F, flow through. Anti-NGF antibody supernatants from FIG. 1were used in this experiment.

FIGS. 6A and 6B show the quantitative purification of anti-canine NGFantibody (HCA isotype) using a three-step method (Method I) comprising(1) anion exchange chromatography, (2) hydrophobic interactionchromatography and (3) size exclusion chromatography. FIG. 6A shows theresults of fractionation by size exclusion HPLC. FIG. 6B shows areducing SDS-PAGE gel of fractions following each step. FIGS. 6C and Dshow the quantitative purification of the anti-canine NGF antibodies ofthe present invention using a two-step method (Method II) comprisingCaptoadhere chromatography and anion exchange chromatography. FIG. 6Cshows SDS-PAGE analysis under non-reducing and reducing conditions. InFIG. 6c , lane 1 is MWS, lane 2 is anti-canine NGF antibody 2 μg/mL and0 μl reducing agent, lane 3 is anti-canine NGF antibody 4 μg/mL and 0 μlreducing agent, lane 4 is anti-canine NGF antibody 6 μg/mL and 0 μlreducing agent, lane 5 is MWS, lane 6 is anti-canine NGF antibody 2μg/mL and 3 μl reducing agent, lane 7 is anti-canine NGF antibody 4μg/mL and 3 μl reducing agent, lane 8 is anti-canine NGF antibody 6μg/mL and 3 μl reducing agent and lane 9 is MWS. FIG. 6D: size exclusionchromatography of the purified anti-canine NGF antibody.

FIG. 7 shows a comparison of anti-canine NGF antibody (HCA isotype)purified by Methods I and II. FIG. 7A: comparison by non-reducing andreducing SDS-PAGE. FIG. 7B: comparison by anti-NGF ELISA.

FIG. 8 shows body weight (upper panel) and temperature (lower panel) arestable following intravenous administration of anti-canine NGFantibodies (HCA isotype, purified by Method I) into dogs.

FIG. 9 shows kinetic analysis of plasma anti-canine NGF monoclonalantibody concentration following intravenous injection to a dog. Abeagle dog was injected intravenously with anti-NGF antibody at 2 mg/kg,samples of plasma were taken at the times indicated and anti-NGFmonoclonal antibody was detected by NGF ELISA. The anti-canine NGFmonoclonal antibody had a surprisingly long elimination (beta) phasehalf life of approximately 9 days.

FIG. 10 shows that anti-canine NGF monoclonal antibodies (HCA isotype,purified by Method I) reduce inflammatory pain in dogs. Kaolin wasinjected into the footpad of beagle dogs at Day −1, antibody or vehiclecontrol at Day 0 and lameness was measured by a visual scoring scale.

DETAILED DESCRIPTION OF THE INVENTION

Following extensive experimentation, the inventor has designed andconstructed several canine monoclonal antibodies using different heavychain constant domain isotypes and has surprisingly shown that usefulproperties can be deduced from their ability (or otherwise) to mediatedownstream effector functions and in particular from their ability tobind to complement. Accordingly, the inventor has identified differentbiological effects mediated by different canine immunoglobulin subtypes.For the complement-binding canine antibody isotypes, this usefulproperty is in directing cells bound by the same antibodies forcomplement directed cytotoxicity (CDC) in-vivo. An example where thisfunctional property is desirable is in canine cancer therapy whereantibodies directed to tumour antigens would then direct the complementsystem to target the cells which have been bound by the antibody, fordestruction.

By contrast, antibody isotypes which do not bind complement and so donot cause CDC are preferred where complement activity is undesirable,for example in the proximity of nerves, in the eye or in alreadyinflamed tissues, or simply due to the desire to reduce the risk of anunforeseen side effect of antibody.

Clearly therefore, the ability to predict which canine isotypes aresuitable for design and use of antibody therapies in canines is a highlydesirable and useful.

Four different canine immunoglobulin G isotypes have been described(calgG-A (canine immunoglobulin G isotype A), calgG-B, calgG-C, andcalgG-D—Tang et al., 2001). For simplicity (and to allow distinction ofdifferent antibody constructions and from light chain components) theheavy chain constant domains are termed HCA (calgG-A), HCB (calgG-B),HCC (calgG-C) and HCD calgG-D) herein.

Purification of Antibodies

A further surprising discovery was made by the inventor in the processof purifying the desirable HCA and HCD isoforms of anti-NGF antibodiesin that neither bound to Protein A, the ligand used at manufacture-scalein industry in the form of Protein A affinity column chromatography toproduce large scale purification of therapeutic proteins. FIG. 5 showsthe relative recovery of HCA, HCB, HCC and HCD isoforms of the anti-NGFantibodies by Protein A affinity chromatography at small scale.Consequently, other methods were needed to purify the HCA or HCDisoforms, as these could not be purified using Protein A affinitychromatography. After extensive experimentation, the inventor hassurprisingly identified two alternative methods (referred to herein asMethod I and Method II) which could be used to purify a caN-HCA-kLCantibody construct, or other canine antibodies which have the HCA or HCDheavy chain isotype.

The first method comprises a combination of anion exchangechromatography, hydrophobic interaction chromatography and sizeexclusion chromatography. The second method comprises a combination ofcaptoadhere affinity chromatography and anion exchange chromatography.FIG. 6 illustrates the purification of the HCA containing anti-NGFantibody by each of the two methods. Highly purified material wasobtained by each method and the two methods produced material withsimilar bioactivity by NGF ELISA (FIG. 7). Since the HCA and HCDisotypes are more similarly related to one another than to HCB and HCCisotypes, the methods are used for both isotypes.

In a further exploration of antibody purification, the inventorsurprisingly found that the aglycosyl HCB* isotype anti-NGF antibody,like the HCB isotype was still able to bind Protein A and so has thedesirable property of lack of CDC activity and purification by Protein Achromatography.

Canine Safety Testing

In order to demonstrate that the antibodies of the present invention,that are designed to have no unwanted CDC activity, are safe to give todogs, the highly purified anti-NGF HCA isotype antibody was injectedinto three dogs by intravenous injection (following prior approval bythe Institutional Animal Ethics Committee—CRL, Ireland). FIG. 8 showsthat in addition to a lack of behavioural changes observed by theveterinarians, the three dogs showed no weight change or pyrexiafollowing injection of HCA isotype antibody (single 2 mg/kg dose).

Plasma kinetics of the HCA isotype antibody in the three dogs wasconsistent with a two-phase distribution and clearance mechanism,including a long beta half-life of approximately 9 days (FIG. 9). Thelack of rapid clearance over the 14 days follow up period was consistentwith their being no anti-antibody response to the canine antibody. Bycontrast human immunoglobulin heavy chain constant domains areimmunogenic in dogs and they are cleared rapidly from the plasma atabout 8 or 9 days post infusion (Richter 1999, Drug Met. Disp. 27,21-25).

Canine Model of Inflammation

All experiments were carried out with prior approval of theInstitutional Ethics Committee (CRL, Ireland). Beagle dogs were injected(=day −1) with kaolin into the footpad of one hind leg in order togenerate a self-resolving inflammation beginning approximately 24 hourslater and which causes the dogs to become temporarily lame. In thismodel, once the initial inflammation response to kaolin recedes, thedogs become steadily less lame over the period of approximately 1-2weeks and then make a full recovery.

Groups of 3 dogs were injected intravenously with either anti-canine(HCA isotype) NGF monoclonal antibodies at 200 μg/kg body weight orphosphate buffered saline as vehicle control (=day 0). The dogs wereassessed for lameness over 7 days by a visual scoring method (score 0,no lameness (full weight bearing); score 1, slight lameness (not fullweight bearing but walking well); score 2, moderate lameness (slightlyweight bearing and not walking well), score 3, severe lameness (notweight bearing)). Observers were blinded to which dogs received whichinjection.

The results are shown in FIG. 10. Lameness scores were reduced in thedogs receiving anti-NGF monoclonal antibodies by day 3 post-injectioncompared with vehicle control, indicating that the anti-NGF monoclonalantibodies had an effect in reducing the pain in the dogs over that seenwith vehicle alone. The delayed activity is consistent with the plasmapharmacokinetics of anti-canine NGF monoclonal antibodies whichdemonstrated a slow tissue distribution (alpha) phase of approximately30 hours and the relatively poor vascularisation of the footpad area.The results shown in FIG. 10 show that the anti-canine NGF antibodies ofthe present invention reduce inflammatory pain in dogs with a consequentreduction in lameness.

Together the results described by this inventor demonstrate thatpurified canine antibodies constructed using the CDC inactive HCAisotype are safe and effective in canines and have a desirable longhalf-life.

All documents referred to in this specification are herein incorporatedby reference. Various modifications and variations to the describedembodiments of the inventions will be apparent to those skilled in theart without departing from the scope of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes of carrying out theinvention which are obvious to those skilled in the art are intended tobe covered by the present invention.

DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the meaning commonly understood by a person who is skilled in theart in the field of the present invention. The meaning and scope of theterms should be clear, however, in the event of any ambiguity,definitions provided herein take precedent over any dictionary orextrinsic definition.

Throughout the specification, unless the context demands otherwise, theterms “comprise” or “include”, or variations such as “comprises” or“comprising”, “includes” or “including” will be understood to imply theinclusion of a stated integer or group of integers, but not theexclusion of any other integer or group of integers.

As used herein, terms such as “a”, an and the include singular andplural referents unless the context clearly demands otherwise. Thus, forexample, reference to “an active agent” or “a pharmacologically activeagent” includes a single active agent as well as two or more differentactive agents in combination, while references to “a carrier” includesmixtures of two or more carriers as well as a single carrier, and thelike. Further, unless otherwise required by context, singular termsshall include pluralities and plural terms shall include the singular.

As herein defined, the term “pain” means an unpleasant sensory andemotional experience associated with actual or potential tissue damage,or described in terms of such damage.

In relation to operative or post-operative pain, the US Animal WelfareAct (Animal Welfare Act 2002. AWA regulations, CFR, Title 9 (Animals andAnimal Products), Chapter 1 (Animal and Plant Health Inspection Service,Department of Agriculture). Subchapter A (Animal Welfare), Parts 1-4)defines a painful procedure as any procedure that would reasonably beexpected to cause more than slight or momentary pain or distress in ahuman being to which that procedure was applied, that is, pain in excessof that caused by injections or other minor procedures. Therefore, if acanine undergoes a painful surgical procedure, the animal should receivepostoperative analgesics.

In further instance, a canine may be experiencing significant or chronicpain as a result of an associated medical condition such as rheumatoidarthritis, osteoarthritis, inflammation or a cancerous or malignantcondition.

The term “nociception” refers to the perception of noxious stimuli. Asherein defined “neuropathic pain” (also known as ‘neuralgia’) is a painthat comes from problems with signals from the nerves. It may arise as aconsequence of a lesion or disease affecting the somatosensory system.There are causes of neuropathic pain and it may be associated withabnormal sensations called dysesthesia, which occur spontaneously.Alternatively, it may be associated with allodynia which results whenthe pain comes on, or gets worse, with a touch or stimulus that wouldnot normally cause pain. For example, a slight touch on the face maytrigger pain if you have trigeminal neuralgia, or the pressure of thebedclothes may trigger pain if you have diabetic neuropathy. Neuropathicpain may also result from allodynia, where the pain comes on, or getsworse, with a touch or stimulus that would not normally cause pain. Forexample, a slight touch to the face may trigger pain if a subject hastrigeminal neuralgia. Neuropathic pain relating to hyperalgesia meansthat severe pain results from a stimulus or touch that would normallycause only slight discomfort, while paraesthesia means thatuncomfortable or painful feelings occur even when there is nothing incontact with the area causing the pain, for example pins and needles.Other forms of neuropathic pain involve pruritis or itch, which can beassociated with allergic or inflammatory responses in the skin andinflammatory pain resulting from tissue damage and repair processes

As defined herein, the term “NGF neutralising antibody” or similardescribes an antibody that is capable of neutralising the biologicalactivation and signalling of NGF. The neutralising antibody, which mayalso be referred to as an antagonistic antibody, or a blocking antibody,specifically, and preferably selectively, binds to NGF and inhibits oneor more biological activities of NGF. For example, the neutralisingantibody may inhibit the binding of a NGF to its target ligand, such asthe cell membrane bound TrkA or p75 receptors.

As used herein, the term “biological activity” refers to any one or moreinherent biological properties of a molecule (whether present naturallyas found in vivo, or provided or enabled by recombinant means).Biological properties include but are not limited to receptor bindingand/or activation; induction of cell signalling or cell proliferation,inhibiting cell growth, induction of cytokine or chemokine production,induction of apoptosis, and enzymatic activity.

The term “complementarity determining region (CDR)”, as used herein,refers to amino acid sequences which together define the bindingaffinity and specificity of the natural Fv region of a nativeimmunoglobulin binding site as delineated by Kabat et al. (Kabat, E. A.,Wu, T. T., Perry, H., Gottesman, K. and Foeller, C. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition. NIH Publication No.91-3242). The term “framework region (FR)”, as used herein, refers toamino acid sequences interposed between CDRs. These portions of theantibody serve to hold the CDRs in appropriate orientation (allows forCDRs to bind antigen).

The term “constant region (CR)” as used herein, refers to the portion ofthe antibody molecule which confers effector functions. In the presentinvention, constant regions typically mean canine constant regions, thatis that the constant regions of the subject canininsed antibodies arederived from canine immunoglobulins.

The term “chimeric antibody” as used herein refers to an antibodycontaining sequences derived from two different antibodies, whichtypically are of different species. Most typically chimeric antibodiescomprise variable domains derived from a donor specifies which bindspecifically to a target epitope and constant domains derived fromantibodies obtained from the target species to whom the antibody is tobe administered.

The term “immunogenicity” as used herein refers to a measure of theability of a targeting protein or therapeutic moiety to elicit an immuneresponse (humoral or cellular) when administered to a recipient. Thepresent invention is concerned with the immunogenicity of the subjectcaninised antibodies. Preferably the antibodies of the present inventionhave no immunogenicity, that is that no neutralising antibodies will beraised against them when administered to a canine, and further, noeffector functions are mediated by the Fc regions of the antibody.

The term “identity” or “sequence identity” as used herein, means that atany particular amino acid residue position in an aligned sequence, theamino acid residue is identical between the aligned sequences. The term“similarity” or “sequence similarity” as used herein, indicates that, atany particular position in the aligned sequences, the amino acid residueis of a similar type between the sequences. For example, leucine may besubstituted for an isoleucine or valine residue. This may be referred toas conservative substitution. Preferably when the amino acid sequencesof the invention are modified by way of conservative substitution of anyof the amino acid residues contained therein, these changes have noeffect on the binding specificity or functional activity of theresulting antibody when compared to the unmodified antibody.

Sequence identity with respect to a (native) polypeptide of theinvention and its functional derivative relates to the percentage ofamino acid residues in the candidate sequence which are identical withthe residues of the corresponding native polypeptide, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercentage homology, and not considering any conservative substitutionsas part of the sequence identity. Neither N- or C-terminal extensions,nor insertions shall be construed as reducing sequence identity orhomology. Methods and computer programs for performing an alignment oftwo or more amino acid sequences and determining their sequence identityor homology are well known to the person skilled in the art. Forexample, the percentage of identity or similarity of 2 amino acidsequences can be readily calculated using algorithms e.g. BLAST(Altschul et al. 1990), FASTA (Pearson & Lipman 1988), or theSmith-Waterman algorithm (Smith & Waterman 1981).

As used herein, reference to an amino acid residue having the “highesthomology” to a second amino acid residue refers to the amino acidresidue which has the most characteristics or properties in common withthe second amino acid residue. In determining whether an amino acidresidue has the highest homology to a second amino acid residue, anassessment may typically be made of factors such as, but not limited to,charge, polarity, hydrophobicity, side arm mass and side arm dimension.

The term “corresponding position” as used herein to refer to an aminoacid residue that is present in a second sequence at a positioncorresponding to a specified amino acid residue in a first sequence isintended to refer to the position in the second sequence which is thesame position as the position in the first sequence when the twosequences are aligned to allow for maximum sequence identity between thetwo sequences. Amino acid residues at corresponding positions have thesame Kabat numbering.

The term “consists essentially of” or “consisting essentially of” asused herein means that a polypeptide may have additional features orelements beyond those described provided that such additional featuresor elements do not materially affect the ability of the antibody orantibody fragment to have binding specificity to canine NGF. That is,the antibody or antibody fragments comprising the polypeptides may haveadditional features or elements that do not interfere with the abilityof the antibody or antibody fragments to bind to canine NGF andantagonise canine NGF functional activity. Such modifications may beintroduced into the amino acid sequence in order to reduce theimmunogenicity of the antibody. For example, a polypeptide consistingessentially of a specified sequence may contain one, two, three, four,five or more additional, deleted or substituted amino acids, at eitherend or at both ends of the sequence provided that these amino acids donot interfere with, inhibit, block or interrupt the role of the antibodyor fragment in binding to canine NGF and sequestering its biologicalfunction. Similarly, a polypeptide molecule which contributes to thecanine NGF antagonistic antibodies of the invention may be chemicallymodified with one or more functional groups provided that suchfunctional groups do not interfere with the ability of the antibody orantibody fragment to bind to canine NGF and antagonise its function.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” means the amount of an agent, binding compound, smallmolecule, fusion protein or peptidomimetic of the invention which isrequired to deliver the required therapeutic effect.

The terms “polypeptide”, “peptide”, or “protein” are usedinterchangeably herein to designate a linear series of amino acidresidues connected one to the other by peptide bonds between thealpha-amino and carboxy groups of adjacent residues. The amino acidresidues are usually in the natural “L” isomeric form. However, residuesin the “D” isomeric form can be substituted for any L-amino acidresidue, as long as the desired functional property is retained by thepolypeptide.

As herein defined an “antibody” encompasses antigen-binding proteinswhich specifically bind to a target antigen of interest, in this casecanine nerve growth factor, having one or more polypeptides that can berecombinantly prepared or which are genetically encodable byimmunoglobulin genes, or fragments of immunoglobulin genes. The term“antibody” encompasses monoclonal and chimeric antibodies, in particularcaninised antibodies, and further encompasses polyclonal antibodies orantibodies of any class or subtype. An “antibody” further extends tohybrid antibodies, bispecific antibodies, heteroantibodies and tofunctional fragments thereof which retain antigen binding.

The phrase “specifically binds to” refers to the binding of an antibodyto a specific protein or target which is present amongst a heterogeneouspopulation of proteins. Hence, when present in specific immunoassayconditions, the antibodies bind to a particular protein, in this casecanine NGF, and do not bind in a significant amount to other proteinspresent in the sample.

As defined herein, a “canine” may also be referred to as a “dog”.Canines can be categorised as belonging to the subspecies with thetrinomial name Canis lupus familiaris (Canis familiaris domesticus) orCanis lupus dingo. Canines include any species of dog and includes bothferal and pet varieties, the latter also being referred to as companionanimals.

The present invention will now be described with reference to thefollowing examples which are provided for the purpose of illustrationand are not intended to be construed as being limiting on the presentinvention. The methods and techniques of the present invention aregenerally performed according to conventional methods well known in theart and as described in various general and more specific referencesthat are cited and discussed throughout the present specification unlessotherwise indicated.

EXAMPLES Example 1 Design and Production of Anti-Canine NGF AntibodiesHaving Different Canine Isotypes

Antibodies directed to canine NGF (termed caN antibodies) were designedand constructed using identical variable heavy domains (VH) joined toheavy chain constant domains (CH2 and CH3) selected from HCA (SEQ IDNO:1), HCB (SEQ ID NO:2), HCC (SEQ ID NO:3) or HCD (SEQ ID NO:4). Avariable light chain (VL) was joined to the canine kappa constant domain(SEQ ID NO:5). The combined amino acid sequences were converted toexpressible form in mammalian cells by the optimal selection of codonsand full chemical gene synthesis and cloning into a mammalian cellexpression vector pcDNA3.1+. Specifically, the designed amino acidsequences were constructed into synthetic cDNA-expressible form andcloned into a mammalian cell expression vector pcDNA3.1(+). Wholeantibody sequences were produced by combining caninised variable domainsequences with C-terminal canine constant heavy or constant light chainsequences. The caninised aD11 VH domain was combined with each of thefour heavy chain isotypes HCA, HCB, HCC and HCD (SEQ ID NO:1-SEQ IDNO:4) and the caninised aD11 VL domain with the canine kappa light chainconstant domain (SEQ ID NO:5). The combined amino acid sequences wereconverted to expressible form in mammalian cells by the optimalselection of codons and full chemical gene synthesis and cloning into amammalian cell expression vector pcDNA3.1+.

Combinations of caninised heavy and light chain cDNA plasmids(caN-HCA-kLC using SEQ ID NO:5 plus SEQ ID NO:1 (HCA); caN-HCB-kLC usingSEQ ID NO:5 plus SEQ ID NO:2 (HCB); caN-HCC-kLC using SEQ ID NO:5 plusSEQ ID NO:3 (HCC) and caN-HCD-kLC using SEQ ID NO:5 plus SEQ ID NO:4(HCD)) were transfected into CHO cells, the supernatants harvested andreacted in ELISA format with NGF. Following incubation and wash steps,the bound canine antibody was detected by reactivity with a goat-anticanine IgG specific polyclonal antibody linked to horseradish peroxidase(HRP) and developed using TMB. The optical density of the resultingproduct was measured at 450 nm and compared with that from mock emptyvector transfected supernatant. The results of binding to NGF for the 4caninised antibody isotypes are shown in FIG. 1. Each of theseantibodies has the same light chain (caN-kLC), this being a light chaincomprising a canine kappa constant domain. Each antibody has a differentheavy chain constant domain. Accordingly a specific heavy chain variabledomain is combined with one of 4 different constant domains (caN-HCA,caN-HCB, caN-HCC or caN-HCD). Equivalent binding to NGF was observed foreach of the canine heavy chain isotypes.

Antibody supernatants were tested for NGF binding by ELISA assay (FIG.1A) and NGF neutralisation by TF-1 cell proliferation inhibition assay(FIG. 1B). As can be seen in FIG. 1, the four isotypes had equivalentactivity to one another in these assays, that is, they all boundspecifically to canine NGF.

Example 2 Complement Deposition Induced by NGF-Captured CaninisedAntibodies

The four antibody-containing supernatants were then assessed for theirability to bind complement when bound to NGF using a complement C1qELISA. Plates were coated with 100 μl/well of 5 μg/ml mouse NGF andblocked with 5% BSA/PBS. Coated wells were incubated for 1 hour at roomtemperature with cell culture supernatants, containing recombinantcaninised anti-NGF IgG isotypes, diluted in PBS/1% BSA (100 μl/well).The plates were washed and incubated for 1 hour at room temperature with100 μl/well of human serum diluted 1/100 in veronal buffered salinecontaining 0.5 mM MgCl₂, 2 mM CaCl₂, 0.05% Tween-20, 0.1% gelatin and0.5% BSA. After washing, plates were incubated with 100 μl of a 1/800dilution of sheep anti-C1q-HRP (Serotec) in PBS/1% BSA. After washing,plates were developed by the addition of 100 μl TMB substrate. Allcomplement C1q binding expressed as A450 minus heat-inactivatedcomplement background. Development was stopped by the addition of 100 μlof 2N H₂SO₄ and absorbance read at 450 nm.

The results are shown in FIG. 2. These results show binding of C1q toimmobilised caninised HCB and HCC type antibodies and no binding of C1qto caninised HCA and HCD type antibodies. Hence, the resultssurprisingly indicate that different canine derived heavy chains exhibitdifferent complement binding and hence activation characteristics andthat the caninised antibodies with type HCA and HCD heavy chainsunexpectedly are preferable for use in antagonising canine NGF.Accordingly, the ability to produce an antibody which binds specificallyto canine NGF, yet which does not mediate CDC is highly advantageous, asan antibody which binds specifically to NGF, yet which mediated animmune response in proximity to the cells expressing NGF, would behighly undesirable.

Example 3 Complement Binding of NGF Captured N-Glycosylated andAglycosyl Variants of Anti-Canine-NGF Monoclonal Antibodies with HCB andHCC Heavy Chain Isotypes

A comparison of the binding of N-glycosylated and aglycosyl variants ofanti-canine-NGF monoclonal antibodies to NGF with HCB and HCC heavychain isotypes was carried out. Expression vectors encoding the lightand heavy chain pairs described by SEQ ID NO:5 and SEQ ID NO:2 (HCB),SEQ ID NO:5 and SEQ ID NO:6 (HCB*), SEQ ID NO:5 and SEQ ID NO:3 (HCC),or SEQ ID NO:5 and SEQ ID NO:7 (HCC*) were co-transfected into CHO cellsand the supernatants compared by binding ELISA to mouse NGF. The resultsare shown in FIG. 3. The left hand panel shows detection by ELISA ofexpression of anti-NGF MAbs constructed with HCB heavy chain (HCB),aglycosyl HCB heavy chain (HCB*), HCC heavy chain (HCC) or aglycosyl HCCheavy chain (HCC*)—the open bars show undiluted supernatant, the shadedbars 1/10 diluted supernatant and C shows an undiluted negative controlsupernatant. Equivalent binding to NGF was observed.

Similarly, antibodies designed to remove the constant domain N-linkedglycosylation site of HCB and HCC (referred to as HCB* (SEQ ID NO: 6)and HCC* (SEQ ID NO:7)) were co-expressed with light chain and assessedfor complement activity (FIG. 3) in an attempt to ablate theircomplement binding. Surprisingly, the aglycosylated HCB* was not capableof binding complement, however the aglycosylated HCC* remained capableof binding complement. The identification of canine derived glycosylatedand aglycosylated heavy chains which do not mediate complement fixing isa particularly advantageous finding as NGF is a soluble mediatorinvolved in nociception.

CHO cell transfectant supernatants from were tested for their ability torecruit complement using the C1q ELISA assay described in Example 2. Theresults are shown in FIG. 3—right hand panel. Together the results inFIG. 3 demonstrate that the ability to recruit complement C1q wasabolished by removal of the N-linked glycosylation site in the B typeheavy chain (HCB*) and was diminished by a similar mutation in the Ctype heavy chain (HCC*).

Accordingly, it is shown herein, quite surprisingly, that where anantibody has a canine-derived heavy chain of the HCA, HCD subtype oraglycosylated HCB* isotype, the binding of the antibody to canine NGFdoes not result in complement activation (and potentially otherdownstream effector functions, such as ADCC and ADCP). Hence, saidantibodies, in antagonising the biological functional activity of atarget, such as canine NGF, by preventing binding of canine NGF to cellmembrane bound TrkA or p75 receptors, inhibit the associated downstreamintracellular signalling cascade. Furthermore, as NGF expressionfrequently occurs in the proximity of nerves, such NGF antagonising orneutralising antibodies, which have canine derived heavy chain of theHCA, HCD or HCB* subtype, sequester canine NGF biological activitywithout recruiting a wider immune response. Hence, the resultssurprisingly indicate that different canine derived heavy chains exhibitdifferent complement binding and activation characteristics and that thecaninised antibodies with type HCA and HCD heavy chains have beenunexpectedly shown to be preferable for use in antagonising canine NGF.The identification of canine derived heavy chains which do not mediatecomplement fixing is a particularly advantageous finding as NGF is asoluble mediator. Such functional properties are both unexpected andhighly desirable.

Example 4 Production of Antibodies with Canine Heavy Chain ConstantDomains to Other Antigens: VEGF and CD20 and their Binding to Complement

Given the surprising result that different canine heavy chain isotypeshave differential binding to complement, anti-VEGF and anti-CD20antibodies were similarly constructed using canine heavy chain constantdomains expressed in CHO cells using the same methodology as describedin Example 1. Assay results from these antibody supernatants in thecomplement ELISA are shown in FIG. 4. The results compared to theanti-NGF antibodies described above for their ability to recruitcomplement following binding to their cognate antigen.

The results are shown in FIG. 4. Panel A shows that caninised anti-NGFMAb constructed using canine heavy chain isotypes B and C and humanisedantibodies constructed using human heavy chain isotypes IgG1 and IgG4were captured onto NGF coated plates, incubated with human serum andbound C1q was detected by ELISA using anti-C1q polyclonal antibodiesconjugated to HRP. Panel B shows the results of complement C1q bindingto VEGF-captured caninised anti-VEGF MAbs constructed using canine heavychain isotypes A and B (SEQ ID NO:8, SEQ ID NO:9). Panel C shows theresults of complement C1q binding to anti-CD20 MAb captured on huCD20extracellular domain peptide (muB-2a: murine anti-human CD20 MAb andmuB-HCB: murine anti-human CD20 MAb expressed as a chimeric fusionprotein with canine heavy chain isotype B (SEQ ID NO:9). All complementC1q binding expressed as A450 minus heat-inactivated complementbackground.

Together these data show that anti-VEGF antibodies constructed using HCBbut not HCA canine heavy chain constant domains could recruit complementand an anti-CD20 antibody constructed using canine HCB could bindcomplement and so parallel the differential binding of anti-NGF antibodyisotypes to complement described above. In summary, these data supportthe surprising observation that antigen-captured antibodies constructedwith HCB and HCC isotypes bind complement whereas HCA and HCD do not.The identification that HCB and HCC isotypes bind complement isparticularly advantageous for tumour cell killing e.g. ofVEGF-expressing or CD20 expressing tumour cells.

The results of these experiments support the unexpected finding fromExample 1 that antibodies comprising canine heavy chain constant domainHCA, HCD and aglycosylated HCB (HCB*) isotypes result in immunoglobulinswhich do not mediate CDC activity. Accordingly, the inventor hasidentified for the first time that such canine derived heavy chains haveutility in immunoglobulins for use in therapeutic methods where a CDCmediated immune response is not desired. Examples of such uses may befound in the inhibition by canine immunoglobulins of cytokines orchemokines, growth factors, hormones and other extracellular mediatorsincluding complement itself in vivo or in diseases such as pain, maculardegeneration or inflammation.

While the HCA, HCD and HCB* isotypes are most useful for design of CDCinactive antibodies, the inventor has also surprisingly identified thatcanine antibodies of HCB (calgG-B) and HCC (calgG-C) isotypes are usefulin the design of CDC active antibodies. Such antibodies are useful whentargeting cells for destruction, for example in cancer therapy. Thereare many human tumour antigens that are targeted using CDC activeantibodies, including CD20, HER2 and the EGFR so canine antibodiesconstructed using HCB and HCC isotypes will have parallel uses incanines.

Example 5 Purification of Anti-NGF Monoclonal Antibodies FollowingExpression in CHO Cells

Since canine anti-NGF monoclonal antibodies of the HCA and HCD isotypeshave desirable lack of binding to complement (FIG. 2), but bind weaklyto Staphylococcus Protein A (FIG. 5), alternative methods ofpurification were developed (FIG. 6). Anti-canine NGF monoclonalantibodies (constructed using heavy chain isotype HCA) were expressed inCHO cells and following extensive experimentation it was surprisinglyfound that the canine anti-NGF antibody could be fractionated to highpurity (>89% monomeric IgG peak, as shown in FIGS. 6A, 6D) by twoalternative purification methods.

In the first method, anti-canine NGF monoclonal antibody was purified byanion exchange chromatography, hydrophobic interaction chromatographyand size exclusion chromatography (Method I—FIGS. 6A and B). In thesecond method, the anti-NGF antibody could be purified by Captoadhereaffinity chromatography followed by anion exchange chromatography(Method II—FIGS. 6C and D).

The main peak of anti-NGF monoclonal antibody purified by either methodcorresponds to a molecular weight of approximately 150 kDa. Comparisonby SDS-PAGE and ELISA (FIG. 7) illustrates that Methods I and II produceantibody preparations with similar purity and bioactivity. Purifiedanti-NGF monoclonal antibodies produced by these methods were tested inthe TF-1 NGF neutralisation assay (described in FIG. 1) and shown tohave high potency (IC50 13 pM anti-NGF neutralised 37 pM NGF; notshown).

Example 6 Anti-Canine NGF Monoclonal Antibodies can be SafelyAdministered Intravenously to Canines and do not Cause Pyrexia

Anti-canine NGF monoclonal antibodies derived from expression vectorscontaining canine HCA type heavy chain were expressed in CHO cells andpurified by a combination of ion exchange chromatography, hydrophobicinteraction chromatography and size exclusion chromatography (Method I,FIGS. 6A and B) and buffer exchanged into phosphate buffered saline. Theantibodies were injected intravenously into beagle dogs at 2 mg/kg bodyweight and assessed for signs of toxicity by visual inspection by aveterinarian, change in body weight, body temperature and plasmabiochemistry. FIG. 8 illustrates the body weight and temperaturemeasurements. No changes were observed in these or any plasmabiochemistry analyte measured (including sodium, potassium, chloride,calcium, phosphate, urea, creatinine, glucose, cholesterol, bilirubin,alanine transaminase, alkaline phosphatase, amylase, lipase, totalprotein or albumin: not shown).

Example 7 Plasma Pharmacokinetics of Anti-Canine (HCA Isotype) NGFMonoclonal Antibodies In-Vivo Demonstrates Long Serum Half-Life and Lackof Immunogenicity

Anti-canine NGF monoclonal antibodies derived from expression vectorsexpressing canine HCA type heavy chain were expressed in CHO cells andpurified by a combination of ion exchange chromatography, hydrophobicinteraction chromatography and size exclusion chromatography and bufferexchanged into phosphate buffered saline (Method 1, FIGS. 6A and B). Theantibodies were injected intravenously into beagle dogs at 2 mg/kg bodyweight and plasma samples were taken at various times over the following2 weeks. Diluted plasma samples were assessed for anti-canine NGFantibody concentration by ELISA using NGF as target and anti-caninepolyclonal antibody-horseradish peroxidase secondary reagent anddeveloped as per FIG. 1. The results are shown in FIG. 9. The plasmaconcentrations measured were consistent with two-phase kinetics, with atissue distribution (alpha) phase half-life of approximately 33 hoursand surprisingly long elimination (beta) phase of approximately 9 days.

The absence of a sharp decline in plasma concentration of anti-canineNGF antibody concentration between 100 and 300 hours demonstrates thatthere are neither pre-existing neutralising antibodies to recombinantanti-NGF monoclonal antibodies in dog blood, nor were any suchneutralising antibodies generated following infusion. By comparison,recombinant human immunoglobulin based proteins are neutralised byantibodies in dog blood at approximately 200 hours post infusion(Richter et al, Drug Metabolism and Disposition 27: 21, 1998). Theseresults therefore show that anti-canine NGF antibodies of the presentinvention have a long serum half-life (approximately 9 days) in vivofollowing intravenous injection and that there are neither pre-existingantibodies nor newly generated antibodies that neutralise the injectedanti-NGF antibodies over time.

Example 8 Effect of Anti-Canine NGF Monoclonal Antibodies in ReducingInflammatory Pain In-Vivo

Antibody Therapy:

Anti-canine NGF monoclonal antibodies derived from expression vectorsincluding canine HCA type heavy chain were expressed in CHO cells andpurified by a combination of ion exchange chromatography, hydrophobicinteraction chromatography and size exclusion chromatography (Method I)and buffer exchanged into phosphate buffered saline.

Canine Model of Inflammation:

All experiments were carried out with prior approval of theInstitutional Ethics Committee (CRL, Ireland). Beagle dogs were injected(=day −1) with kaolin into the footpad of one hind leg in order togenerate a self-resolving inflammation beginning approximately 24 hourslater and which causes the dogs to become temporarily lame. In thismodel, once the initial inflammation response to kaolin recedes, thedogs become steadily less lame over the period of approximately 1-2weeks and then make a full recovery.

Groups of 3 dogs were injected intravenously with either anti-canine NGFmonoclonal antibodies at 200 μg/kg body weight or phosphate bufferedsaline as vehicle control (=day 0). The dogs were assessed for lamenessover 7 days by a visual scoring method (score 0, no lameness (fullweight bearing); score 1, slight lameness (not full weight bearing butwalking well); score 2, moderate lameness (slightly weight bearing andnot walking well), score 3, severe lameness (not weight bearing)).Observers were blinded to which dogs received which injection.

The results are shown in FIG. 10. Lameness scores were reduced in thedogs receiving anti-NGF monoclonal antibodies by day 3 post-injectioncompared with vehicle control, indicating that the anti-NGF monoclonalantibodies had an effect in reducing the pain in the dogs over that seenwith vehicle alone. The delayed activity is consistent with the plasmapharmacokinetics of anti-canine NGF monoclonal antibodies whichdemonstrated a slow tissue distribution (alpha) phase of approximately30 hours and the relatively poor vascularisation of the footpad area.The results shown in FIG. 10 show that the anti-canine NGF antibodies ofthe present invention reduce inflammatory pain in dogs with a consequentreduction in lameness.

All documents referred to in this specification are herein incorporatedby reference. Various modifications and variations to the describedembodiments of the inventions will be apparent to those skilled in theart without departing from the scope of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes of carrying out theinvention which are obvious to those skilled in the art are intended tobe covered by the present invention.

The invention claimed is:
 1. A method of treating a canine to neutralizea target antigen without inducing complement dependent cytotoxicity,comprising administering to a canine subject in need thereof atherapeutically effective amount of an antibody, fusion protein orantigen-binding fragment of an antibody that specifically binds thetarget antigen, wherein said antibody, fusion protein, orantigen-binding fragment comprises a heavy chain constant domaincomprising the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 11; SEQID NO: 13, wherein the heavy chain constant domain does not bind C1qwhen the antibody, fusion protein, or antigen-binding fragment is boundto the target antigen.
 2. The method as claimed in claim 1, wherein themethod is for treating, inhibiting or ameliorating pain or inflammationin the canine subject.
 3. The method as claimed in claim 2, wherein thepain is selected from the group consisting of neuropathic pain,oncologic pain, pain associated with, or resulting from, rheumatoidarthritis, pain associated with, or resulting from, osteoarthritis, painassociated with, or resulting from, inflammation and pain associatedwith, or resulting from, pruritis.
 4. The method as claimed in claim 1,wherein the method minimizes activation of one or more downstream immunesystem effector functions selected from the group consisting of antibodydependent cell mediated cytotoxicity and antibody dependent cellularpathogenesis.
 5. The method as claimed in claim 1, wherein the antibody,fusion protein or binding fragment has a heavy chain constant domaincomprising the amino acid sequence of SEQ ID NO:
 13. 6. A method oftreating a canine to induce destruction of a target displaying a targetantigen through complement dependent cytotoxicity, comprisingadministering to a canine subject in need thereof a therapeuticallyeffective amount of an antibody, fusion protein or antigen-bindingfragment of an antibody that that specifically binds the target antigen,wherein said antibody, fusion protein, or antigen-binding fragmentcomprises a heavy chain constant domain comprising the amino acidsequence of SEQ ID NO: 9, SEQ ID NO: 10; SEQ ID NO: 14, wherein theheavy chain constant domain binds C1q when the antibody, fusion protein,or antigen-binding fragment is bound to the target antigen.
 7. Themethod as claimed in claim 6, wherein the method is for the treatment orprevention of a cancerous or malignant condition in the canine subject.8. The method as claimed in claim 6, wherein the method mediatesactivation of one or more downstream immune system effector functionselected from the group consisting of antibody dependent cell mediatedcytotoxicity and antibody dependent cellular pathogenesis.
 9. The methodas claimed in claim 6, wherein the target antigen is a cancer specificantigen.
 10. The method as claimed in claim 9, wherein the cancerspecific antigen is selected from the group consisting of a cytokine, achemokine, a growth factor, a cell surface receptor, a virus and acomponent of the complement cascade.
 11. The method as claimed in claim9, wherein the cancer specific antigen is selected from the groupconsisting of proteins CD2, CD4, CD8, CD20, EGFR, VEGFR and HER2.